Establishment of prostate cancer spheres from a prostate cancer cell line after phenethyl isothiocyanate treatment and discovery of androgen-dependent reversible differentiation between sphere and neuroendocrine cells.

Prostate cancer can transform from androgen-responsive to an androgen-independent phenotype. The mechanism responsible for the transformation remains unclear. We studied the effects of an epigenetic modulator, phenethyl isothiocyanate (PEITC), on the androgen-responsive LNCaP cells. After treatment with PEITC, floating spheres were formed with characteristics of prostate cancer stem cells (PCSC). These spheres were capable of self-renewal in media with and without androgen. They have been maintained in both types of media as long term cultures. Upon androgen deprivation, the adherent spheres differentiated to neuroendocrine cells (NEC) with decreased proliferation, expression of androgen receptor, and PSA. NEC reverse differentiated to spheres when androgen was replenished. The sphere cells expressed surface marker CD44 and had enhanced histone H3K4 acetylation, DNMT1 down-regulation and GSTP1 activation. We hypothesize that PEITC-mediated alteration in epigenomics of LNCaP cells may give rise to sphere cells, whereas reversible androgenomic alterations govern the shuttling between sphere PCSC and progeny NEC. Our findings identify unrecognized properties of prostate cancer sphere cells with multi-potential plasticity. This system will facilitate development of novel therapeutic agents and allow further exploration into epigenomics and androgenomics governing the transformation to hormone refractory prostate cancer.

Hypoacetylation, hypomethylation, and dephosphorylation of H2B histones and excessive histone deacetylase activity in DU-145 prostate cancer cells.

BACKGROUND: Hypoacetylation on histone H3 of human prostate cancer cells has been described. Little is known about the modifications of other histones from prostate cancer cells. METHODS: Histones were isolated from the prostate cancer cell line DU-145 and the non-malignant prostatic cell line RC170N/h. Post-translational modifications of histone H2B were determined by liquid chromatography-mass spectrometry (LC-MS)/MS. RESULTS: The histone H2B of the prostate cancer cell line DU-145 was found to have hypoacetylation, hypomethylation, and dephosphorylation as compared to the non-malignant prostatic cell line RC170N/h. H2B regained acetylation on multiple lysine residues, phosphorylation on Thr19, and methylation on Lys23 and Lys43 in the DU-145 cells after sodium butyrate treatment. CONCLUSIONS: The histone H2B of DU-145 prostate cancer cells are hypoacetylated, hypomethylated, and dephosphorylated. Histone deacetylase inhibitor reversed this phenotype. Epigenetic agent may therefore be useful for prostate cancer therapy and worth further investigation.

Interaction of a small molecule Natura-α and STAT3-SH2 domain to block Y705 phosphorylation and inhibit lupus nephritis.

A small molecule, Natura-α, a clinical stage investigational new drug for certain inflammatory diseases, has been evaluated for drug interaction with STAT3 and inhibiting systemic lupus erythematosus (SLE). Studies have revealed that it selectively inhibits STAT3-Y705 phosphorylation and, suppresses pro-inflammatory cytokines, stimulates anti-inflammatory cytokine IL-10, thereby skewing T cell differentiation from the Th1/Th17 lineages toward the Treg lineage. The potential binding of the drug to STAT3 protein has been investigated with a computational modeling and docking simulation using X-ray crystal structure of the STAT3ß homodimer. Natura-α was shown to directly bind to SH2 domain of STAT3 and forms H-bonds with amino acids Glu594 and Arg609. The phosphorylation of Y705 was prevented and making the formation of STAT3 homodimer impossible, thereby blocking STAT3 activation. The in vivo efficacy of Natura-α in SLE was evaluated in a bioassay with NZB/W female mice. Mice at week 19 were given orally Natura-α at 25 or 75 mg/kg, once a day, 5 days per week for 29 weeks. Mice were monitored weekly until 52 weeks of age. Both dosages were effective to reduce proteinuria and significantly improved animal survival rate. The renal functions were preserved with glomerular lesions reversed, which paralleled with decreased C3 deposit. The numbers of kidney cells stained with phosphorylated STAT3-Y705 remarkably decreased, demonstrating blocking of Y-705 phosphorylation by the treatment. Since NZB/W mice develop nephritis which resembles SLE in men, the data strongly suggests that Natura-α may be a potential effective therapeutic agent for lupus.

Prostate cancer chemopreventive activity of phenethyl isothiocyanate through epigenetic regulation (review).

Prostate cancer is one of the most commonly diagnosed cancers in men. The number of affected men is expected to rapidly increase as the population of males over the age of 50 grows worldwide. For patients who are not cured by local treatment and experience metastatic disease, neither androgen ablation nor chemotherapy can abrogate progression and death from androgen-independent/hormone-refractory disease. Therefore, finding strategies for the prevention of prostate cancer initiation and disease progression is a medical challenge. Consumption of cruciferous vegetables has been reported to be associated with reduced incidence of prostate cancer cases. The isothiocyanates, including phenethyl isothiocyanate (PEITC), from cruciferous vegetables have been demonstrated as active components responsible for chemoprevention. In this review, we summarize the recent findings of PEITC on prostate cancer prevention with an emphasis on epigenetic mechanisms. Studies have indicated that PEITC mediates gene regulation, such as downregulation of androgen receptor expression and induction of endogenous cyclin-dependent kinase inhibitors, p21 and p27. The gene for detoxifying enzyme pi-class glutathione S-transferase (GSTP1), silenced in the vast majority of prostate tumor cells, could be reactivated and the enzymatic function recovered. This may be through epigenetic mechanisms as PEITC is a dual inhibitor of histone deacetylases and aberrant CpG island methylation of various genes. The epigenetic regulation may play a critical role, along with interactive mechanisms including the disruption of microtubule polymerization, in prostate cancer prevention by PEITC. These mechanisms target and correct the aberrations fundamental to the initiation and progression of carcinogenesis in cells, and restoring the cells to a more normal state. Inhibiting and eliminating cancer cells forms the basis of cancer prevention.

Deficient histone acetylation in acute leukemia and the correction by an isothiocyanate.

OBJECTIVE: Since histone hypoacetylation due to excess histone deacetylases (HDACs) has been associated with transcriptional repression in leukemia, we aimed to determine deficient histone acetylation in patients with acute leukemia and the effect of its correction by an isothiocyanate. METHODS: The acetylation status of histones H3 and H4 in cells from patients with untreated acute leukemia was determined by Western blot. Deficient histone acetylation was analyzed in relation to the disease state. Bone marrow cells from 10 patients with acute myeloid leukemia (AML) were cultured in phenylhexyl isothiocyanate (PHI) to evaluate correction of the deficiency. RESULTS: Acetylation of histones H3 and H4 was virtually undetectable or significantly lower in acute leukemia. This deficiency was consistent among all the patients examined. Histone acetylation was up-regulated in the presence of PHI, revealing an excess of deacetylation activity in AML. PHI treatment induced apoptosis, indicating HDAC inhibition was able to correct the deficiency. CONCLUSIONS: Deficient histone acetylation may represent an aberration at the epigenetic level in acute leukemia. PHI might represent a target for correcting deficient acetylation, and potential epigenetic regulators for preventing the progression of leukemia.

Deficient histone acetylation and excessive deacetylase activity as epigenomic marks of prostate cancer cells.

Aberrant epigenomic alterations include incorrect histone modifications involving altered expression of chromatin-modifying proteins. They contribute to gene silencing and carcinogenesis. The nature of the epigenomic alterations occurring with prostate cancer remains to be fully identified. The acetylation status of histone H3 in human prostate cancer cells was assessed with multiple acetylation sites at N-termini. In contrast to the non-malignant prostatic cell lines RC165N/h and RC170N/h which possess stem cell properties, cancer cell lines LNCaP, DU-145, and PC-3 were either not acetylated or reduced in density (50-70%), at N-termini lysines 9, 14, 18, and 23 of histone H3. Deficient acetylation of histone H3 was similarly detected with clinical prostatic adenocarcinomas as compared to normal tissues. Cancer cell lines and adenocarcinomas exhibited varied acetylation status at particular lysines, indicating the possible presence of deacetylation patterns reflecting individual cancer cell clones. A significantly elevated activity of histone deacetylases (HDACs) was determined in both cancer cell lines and adenocarcinomas. Inhibition of HDACs enhanced histone acetylation and p21 gene expression, indicating that excessive HDAC activity is a requisite for deficient histone acetylation. Deficient histone acetylation involving excessive HDAC activity may represent epigenomic features of prostate cancer cells, and the aberrant enzyme activity is probably an underlying cause of disrupting the epigenomes of normal prostatic cells.

De-repression of the p21 promoter in prostate cancer cells by an isothiocyanate via inhibition of HDACs and c-Myc.

Natural isothiocyanates from cruciferous vegetables have been described as important dietary factors for prostate cancer prevention. Phenethyl isothiocyanate (PEITC), found rich in watercress, induces growth arrest and apoptosis in prostate cancer cells, and also inhibits the testosterone-mediated growth of prostates by regulating the androgen receptor and cell cycle progression in rats. PEITC has been recently identified as an inhibitor of histone deacetylases (HDACs). Herein we describe the mechanism of PEITC-mediated growth attenuation in relation to HDAC inhibition in human prostate cancer cells. Exposure of androgen-dependent prostate cancer cells LNCaP to PEITC resulted in cell cycle arrest and a p53-independent up-regulation of the inhibitors of cyclin-dependent kinases, including p21WAF1 and p27. The mechanism of p21 activation was investigated. PEITC significantly enhanced histone acetylation and induced selective modification of histone methylation for chromatin remodeling. Chromatin immunoprecipitation revealed that the p21 gene was associated with the PEITC-induced hyperacetylated histones. As a result, the chromatin unfolding permitted the transcription activation of the p21 gene. PEITC also significantly reduced the expression of c-Myc which represses p21. Pull-down assays using Sp1 affinity oligo beads of the p21 promoter, showed decreased c-Myc binding to the Sp1 transcriptional complexes in the p21 promoter, resulting in reduced p21 repression. The quantity of PEITC (0.5-1 micro M) effective to mediate cell cycle arrest was less than that for inhibiting c-Myc (2-5 micro M), suggesting that the inhibition of HDACs may be the primary mechanism for p21 activation. The PEITC-mediated growth attenuation of prostate cancer cells includes an interactive mechanism involving HDAC and c-Myc inhibition.

Modulating testosterone stimulated prostate growth by phenethyl isothiocyanate via Sp1 and androgen receptor down-regulation.

BACKGROUND: The effects of phenethyl isothiocyanate (PEITC), present naturally in cruciferous vegetables, on androgen-influenced growth of the prostate such as benign hyperplasia, was investigated. METHODS: Rats dosed with cyproterone acetate and testosterone, were fed at the same time with either PEITC or vehicle control. The growth of the prostates was compared to untreated rats. RESULTS: While testosterone increased the prostate mass (30%) and hyperplastic seminiferous tubules as compared to the untreated rats, PEITC feeding decreased the prostate mass and hyperplasia to roughly the levels of untreated rats (P < 0.05). PEITC negated the testosterone-mediated enhancement of the androgen receptor (AR), via down-regulating transcription factor Sp1 expression and Sp1 binding complex formation. Cell cycle progression was attenuated with decreases of cyclins, Rb, and up-regulates p27. CONCLUSIONS: PEITC modulates the testosterone-influenced growth by repressing Sp1, thus down-regulating AR and proliferation. PEITC from cruciferous vegetables may represent a regulator for hormone-dependent growth of the prostate.

Dual action on promoter demethylation and chromatin by an isothiocyanate restored GSTP1 silenced in prostate cancer.

Prostate carcinoma is characterized by the silencing of pi-class glutathione S-transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1, due to aberrant methylation at the CpG island in the promoter/5'-UTR, occurs in the vast majority of prostate tumors and precancerous lesions. It is a pathologic marker and probably an underlying cause of oxidative damage and inflammation at tumor initiation. Inhibition of the aberrant promoter methylation could therefore be an effective mean to prevent carcinogenesis. Several isothiocyanates, including phenethyl isothiocyanate (PEITC), found naturally in cruciferous vegetables, induced growth arrest and apoptosis in prostate cancer cells in culture and xenografts. The effects of PEITC to reactivate GSTP1 were investigated. Exposure of prostate cancer LNCaP cells to PEITC inhibited the activity and level of histone deacetylases (HDACs), and induced selective histone acetylation and methylation for chromatin unfolding. Concurrently PEITC demethylated the promoter and restored the unmethylated GSTP1 in both androgen-dependent and -independent LNCaP cancer cells to the level found in normal prostatic cells, as quantified by methylation-specific PCR and pyrosequencing. The dual action of PEITC on both the DNA and chromatin was more effective than 5'-Aza-2'-deoxycytidine, sodium butyrate, or trichostatin A (TSA), and may de-repress the methyl-binding domain (MBD) on gene transcription. The PEITC-mediated cross-talk between the DNA and chromatin in demethylating and reactivating GSTP1 genes, which is critically inactivated in prostate carcinogenesis, underlines a primary mechanism of cancer chemoprevention. Consequently, new approaches could be developed, with isothiocyanates to prevent and inhibit malignancies.

The phenylhexyl isothiocyanate induces apoptosis and inhibits leukemia cell growth in vivo.

Isothiocyanates are potent chemopreventive agents for carcinogen-induced cancers in rodents. The major mode of action for chemoprevention is cytoprotection i.e. inducing detoxifying enzymes to remove the carcinogens, thus blocking the initiation of carcinogenesis. Analysis has indicated that isothiocyanates also act at the post-initiation levels of carcinogenesis. We have also reported that the phenylhexyl isothiocyanate (PHI) induced growth arrest and apoptosis in human leukemia HL-60 cells in culture. Since then we have investigated the in vivo efficacy of PHI. The effects of PHI were evaluated in immunodeficient mice, with xenografts of human leukemia HL-60 cells. The maximum tolerated dose (MTD) was determined. The experimental mice received 80% of the MTD. Oral feedings of PHI significantly reduced tumor incidence (p<0.05) without overt toxicity. PHI inhibited cell cycle progression through the down-regulation of cyclin expression, Rb phosphorylation and the up-regulation of the cdk-inhibitors. Apoptosis was significantly increased in the treated tumors but not in the normal mouse tissues. In conclusion, PHI induced apoptosis and inhibited the growth of xenografts by targeting the cell cycle regulators. PHI induced selective apoptosis effects in the rapidly growing tumor cells but not in the normal tissues.

Repression of androgen receptor in prostate cancer cells by phenethyl isothiocyanate.

BACKGROUND: Prostate cancer usually progresses to androgen refractory after an initial anti-androgen treatment. The androgen receptor (AR) is a pivotal factor for the androgen-mediated growth and maintenance of the prostate. Abnormality of the AR, such as overexpression has been postulated to be related to the hormone independent growth of the cancer. Although we previously demonstrated that the AR expression could be modulated by isothiocyanates, which are natural constituents of cruciferous vegetables, the mechanism, however, remained to be clarified. We have since investigated the mechanism of phenethyl isothiocyanate (PEITC) in AR regulation. METHODS: A human androgen dependent prostate cancer cell line LNCaP (AD) and its sub-line LNCaP (AI), i.e. androgen independent but overexpressing AR, were exposed to PEITC. The effects of PEITC on cell growth and AR expression/transcription were analyzed with MTT assay, real-time PCR and western blotting. The AR promoter activity was analyzed with the reporter activity after transfection with pAR-luc. The effects on Sp1, the major transcription factor of the AR, were tested with Sp1-luc activity, western blotting and electrophoretic mobility shift assay. RESULTS: PEITC induced a significant growth inhibition, with equal IC(50), in both AD and AI cells. The AR present in both cells was repressed as demonstrated with real-time PCR and western blot. PEITC mediates dual effects at transcriptional and post-translational levels to regulate the AR. At transcriptional level the AR level was reduced via inhibition of the transcription factor Sp1, and at post-translational level by accelerating protein degradation. CONCLUSION: PEITC represses AR transcription and expression, and mediates growth arrest in androgen dependent and independent prostate cancer cells. With the AR modulation and growth attenuation, PEITC and possibly other isothiocyanates, may prevent and inhibit hormone sensitive and refractory prostate cancer.

Phenylhexyl isothiocyanate inhibits histone deacetylases and remodels chromatins to induce growth arrest in human leukemia cells.

Natural isothiocyanates, present in cruciferous vegetables and synthetic phenylhexyl isothiocyanate (PHI) are chemopreventive agents which act by blocking the initiation of carcinogen-induced tumors in rodents. We have demonstrated that isothiocyanates are also growth regulators, inhibiting cell cycle cdk activity and up-regulating inhibitor p21WAF1 (p21) in cancer cells. The up-stream mechanism to modulate cell cycle progression remained to be elucidated. Here, we have demonstrated that exposure of HL-60 leukemia cells to PHI induced G1 arrest and apoptosis. The hypothesis that PHI inhibits cell growth via chromatin remodeling was investigated. PHI mediates the complex cross talk between chromatin and DNA, and it was demonstrated for the first time as an inhibitor of histone deacetylases (HDAC). Thus, the HDAC activity in PHI-exposed HL-60 cells was reduced. Additionally, PHI reduced the expression of HDAC and increased the level of acetyl transferase p300, in favor of accumulation of acetylated histones. Within hours, global acetylation of histones was enhanced. PHI further mediated selective alterations of histone methylation, with a pattern consistent to the marks of transcription competent chromatins. The relationship between acetylated histones and p21 was examined by chromatin immunoprecipitation (ChIP) assay. Chromatins from cells exposed to PHI contained more p21 DNA in the precipitates of hyperacetylated histones, indicating more accessibility of transcription machinery to the p21 promoter after chromatin unfolding. The cell cycle inhibitors were activated as a result. In contrast to the PHI-induced apoptosis in HL-60 cells, which was mediated by caspase-9 up-regulation and bcl-2 reduction, PHI did not induce significant apoptosis in the mononuclear cells from normal peripheral blood and bone marrow. The results revealed a potential selective effect of isothiocyanates to inhibit the growth of malignant cells.

Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells.

The relation between the consumption of cruciferous vegetables and reduced prostate cancer occurrence has been documented, although the responsible phytochemicals are unknown. The effects of sulforaphane (SFN) which occurs as the precursor glucosinolate in broccoli and other cruciferous vegetables, and its metabolite N-acetylcysteine conjugate (SFN-NAC) on prostate cancer cells were investigated. SFN and SFN-NAC were analyzed with the androgen-dependent human prostate cancer LNCaP cell line model. Cell growth and apoptosis were determined with the expression of androgen receptor and prostate specific antigen, DNA synthesis, cell cycle progression, DNA strand breaks and caspase activation to ascertain the effects and mechanism. SFN and SFN-NAC were demonstrated for the first time to mediate a dose-dependent apoptosis and growth arrest in the prostate cancer cells. Caspases were activated and DNA strand breaks were detected in apoptotic cells. The expression of phosphorylated and dephosphorylated androgen receptors, and the production of prostate specific antigen were attenuated. The expression of cyclin D1 and DNA synthesis were inhibited along with G1 cell cycle block, causing decreased cell density and growth. SFN and its metabolite SFN-NAC have similar activities to induce growth arrest and apoptosis, indicating that the effects of SFN are maintained through the metabolic processes. SFN as a dietary component of cruciferous vegetables active in the prevention of prostate cancer is discussed.

Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis.

Recent studies in cell culture have shown that isothiocyanates (ITCs) induce apoptosis via activation of mitogen-activated protein (MAP) kinases and p53 pathways, suggesting a potential for ITCs or their conjugates to inhibit tumorigenesis during the postinitiation phase. To evaluate whether ITC compounds administered after carcinogen treatment inhibit lung tumorigenesis, we investigated in A/J mice the effects of the N-acetylcysteine (NAC) conjugates of benzyl (BITC-NAC) and phenethyl ITC (PEITC-NAC) in the diet (15 micromol/g) administered after a single dose of 20 micromol benzo(a)pyrene [B(a)P]. The formation of lung adenomas was examined 140 days after B(a)P dosing. Both the BITC-NAC and PEITC-NAC-treated groups showed a significant reduction in lung tumor multiplicity from 6.1 +/- 3.1 tumors/mouse in the B(a)P group fed the control diet to 3.7 +/- 2.9 and 3.4 +/- 2.7 tumors/mouse (P = 0.018 and 0.006, respectively). To investigate the mechanisms of tumor inhibition, lung tissues were obtained at 21, 84, and 140 days at interim sacrifices during the bioassay. These tissues showed a significant increase in apoptosis as determined by in situ end-labeling for both ITC-NAC-treated groups. The MAP kinase pathway was activated in the ITC-NAC-treated groups. The activation of c-Jun NH(2)-terminal kinase was higher in the BITC-NAC and PEITC-NAC groups when compared with B(a)P-treated control. The phosphorylation of p38 and extracellular signal-regulated kinases (ErKs) 1 and 2 was also induced by these treatments. To determine the downstream target of MAP kinases, activator protein-1 (AP-1) and nuclear factor-kappaB activities were evaluated by gel shift assay. The AP-1 binding activity was remarkably increased in lung tissue from both the BITC-NAC and PEITC-NAC groups. No change in nuclear factor-kappaB binding activity was found, however. Phosphorylation of p53 was also higher than the constitutive levels in both ITC-NAC-treated groups, but no induction of p53 expression was detected. This study demonstrates the chemopreventive efficacy of the NAC conjugates of PEITC and BITC administered in the diet after a single dose of B(a)P for lung tumorigenesis and provides the first in vivo evidence that activation of MAP kinases, AP-1 transcription factors, p53 phosphorylation, and the induction of apoptosis may be involved in the chemopreventive activity of these compounds.

Endothelin-1 from prostate cancer cells is enhanced by bone contact which blocks osteoclastic bone resorption.

The causes for the propensity of metastasized prostate cancer cells to grow in bone and to induce osteoblastic lesions remain unresolved. Co-culture of human prostate cancer cell lines with bone slices was determined to increase the level of endothelin-1 (ET-1) mRNA and its production. ET-1 is an ejaculate protein that also stimulates osteoblasts. Osteoclastic bone resorption was significantly blocked by the presence of androgen-independent prostate cancer cells in a dose-dependent manner as that of synthetic ET-1. The inhibition could be neutralized by specific ET-1 antibody, indicating the association of prostate cancer-derived ET-1 with inhibition of bone resorption. The combined ET-1 activity on osteoclasts and osteoblasts disrupts bone remodelling. ET-1 production is also elevated in the presence of prostate-specific antigen (PSA). ET-1 in turn enhances DNA synthesis of prostate cancer cells. Interactions among cancer cells, bone, ET-1 and PSA may be critical in cancer growth and lesions in bone.

Modulation of lymphocyte proliferative responses by a canine Lyme disease vaccine of recombinant outer surface protein A (OspA).

The modulation of human lymphocyte proliferative responses was demonstrated with a recombinant outer surface protein A (OspA) vaccine preparation for the prevention of Borrelia burgdorferi infection. After exposure to either the unaltered vaccine preparation or OspA prepared in saline, normal lymphocyte responses to the mitogens concanavalin A, phytohemagglutinin-M or pokeweed mitogen, or the antigen BCG were consistently reduced. Whole cell extracts of B. burgdorferi also modulated immune responses but required a much greater quantity of protein than needed for the OspA preparation. The magnitude of modulation was directly dependent on the quantity of OspA. OspA interferes with the response of lymphocytes to proliferative stimuli including a blocking of cell cycle phase progression. Future studies designed to delete the particular region or component of the OspA molecule responsible for this effect may lead to improved vaccine preparations.

Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate.

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.

Cyclosporine-induced autologous graft-versus-host disease in patients with acute myeloid leukemia undergoing non-myeloablative chemotherapy without progenitor cell reinfusion.

To determine the incidence and severity of cyclosporine-induced graft-versus-host disease following non-myeloablative chemotherapy without progenitor cell reinfusion in patients with acute leukemia, 17 adults with refractory acute myeloid leukemia (14) or blastic phase of chronic myeloid leukemia (3) were treated with etoposide 2400 mg/m2 and cyclophosphamide 120 mg/kg followed by cyclosporine (CsA) 2.5 mg/kg i.v. daily and interferon gamma 0.025 mg/m2 subcutaneously every other day until day 28. Skin biopsies were obtained on days 14 and 28, or on the appearance of a skin rash, and graded for GVHD. Blood samples were examined at baseline and weekly starting on day 14 for natural killer (NK) cell and T cell lymphocytic changes. Post-treatment lymphocytes from select patients were assessed for allogeneic NK cell and autologous leukemic cell cytolytic activity. Four patients developed pathologic grade 2 cutaneous acute GVHD. Of the three patients who achieved a complete remission, two had evidence of GVHD. Post-treatment, three patients (two with GVHD) in whom adequate numbers of lymphocytes could be obtained showed NK cell cytolytic activity against allogeneic tumor cells (K562), but none had cytolytic activity against their own cryopreserved leukemic cells. These data suggest that in patients with AML treated with subablative doses of chemotherapy without autotransplant, autologous GVHD can be induced, although at an incidence lower than that reported for CsA-induced GVHD following marrow transplantation. An enhancement of T cell and NK cell activity levels similar to experiences in syngeneic models of autologous GVHD was seen, but no direct autologous leukemic cell cytotoxicity could be demonstrated.

Development of human prostate cancer cells to neuroendocrine-like cells by interleukin-1.

Growth and development of some prostate epithelial cells are androgen-dependent. Non-androgenic hormones and growth factors may also influence prostate cells and the effect of interleukin 1beta (IL-1beta) has been investigated with an androgen-dependent human prostate cancer cell line LNCaP. Exposure of LNCaP cells to IL-1beta at picomole ranges resulted in a dose-dependent and progressive differentiation to neuroendocrine-like cells evidenced by dendrite formation and the development of specific neuroendocrine cell markers. Quantification by computer-based image analysis after immunostaining revealed a two-fold increase of chromogranin A in 90% of the cells and a ten-fold increase in the remaining 10%. Additionally, serotonin was developed in all the cells with the staining intensity increased by five-fold. Significant increase in cytokeratin 8 and reduction of prostate specific antigen was also noted. Proliferation was reduced in parallel to the cellular development. The IL-1beta effect was irreversible after several days of IL-1beta incubation. IL-1beta is produced constitutively and its secreted level has an inversed relation during the exponential and plateau phases of cell growth. An IL-1 autocrine regulation in the growth and differentiation of prostate cells is discussed.

Regulation of growth and apoptosis of breast cancer cells by a 54 kDa lymphokine.

A normal human lymphocyte-derived 54 kDa polypeptide, capable of regulating cell growth has been identified as an isoform variant (abbreviated as NP54) of protein neuroleukin-phosphoglucose isomerase (PGI). Since distinct PGI variants undetectable in normal tissues had been identified in breast cancer tissues, the effect of NP54 on the growth of human breast cancer cells SK-Br-3 in cultures was analyzed. Exposure to NP54 caused a dose-dependent growth modulation. Approximately 40% reduction of cell density was detected at 40 pM of NP54, along with a blocking of G1 cells entering into S phase. The growth modulation was correlated with a significantly reduced expression of epidermal growth factor receptor (EGF-R) gene transcript, supporting the interpretation that the level of EGF-R expression and cell growth are related mechanisms. NP54 treatment also significantly increased cells with apoptotic morphological feature and fragmented DNA. Incubation with a monoclonal anti-NP54 antibody negated NP54 activity, confirming a regulatory activity in cell growth and apoptosis.